The levels of plasma high density lipoprotein (HDL) are inversely related to the incidence of atherosclerotic cardiovascular disease. One explanation for this relationship is that low levels of HDL cholesteryl esters (CE) indicate an accelerated rate of transfer of CE from HDL to atherogenic apoB-containing lipoproteins. The transfer is mediated by a plasma CE transfer protein (CETP). The objective of the proposal is to study the structure and mechanism of action of the CETP molecule and its role in HDL metabolism. Using available specific antibodies and aminoacid sequence information, a liver cDNA library will be screened in order to obtain a cDNA to the CETP. The cDNA will be used to derive the complete aminoacid sequence of CETP and to examine its expression in different tissues and the regulation of its synthesis in HepG2 cells. To evaluate the possibility that the CETP acts as a carrier of neutral lipids, its ability to bind CE and to transport CE between lipoproteins or phospholipid monolayers will be examined. Certain proteolytic fragments of CETP have been found to promote CE transfer. The active portion of the CETP molecule will be defined by peptide isolation and sequencing, by labeling with photoactive CE derivatives, by immunoblotting with inhibitory monoclonal antibodies and by expression of parts of the cDNA of CETP in eukaryotic cells or bacteria. In human studies the rate of transfer of CE from HDL to apoB-containing lipoproteins will be measured in fasting or alimentary lipemic plasma in order to evaluate the hypothesis that normolipidemic subjects with lower HDL CE levels have more rapid CETP- mediated CE transfer. These studies will provide important basic information on the CETP molecule and will help to define its role in lipoprotein and atherogenesis.